脊柱结核椎间盘LncRNA表达差异性分析及潜在诊断标志物筛选

Analysis of differential expression LncRNA in intervertebral discs of spinal tuberculosis and screening of potential diagnostic markers

中文关键词:  脊柱结核  长链非编码RNA  诊断标志物

英文关键词:Spinal tuberculosis  LncRNA  Diagnostic markers

中文摘要:

  【摘要】 目的：应用表达谱芯片检测脊柱结核（spinal tuberculosis，STB）患者椎间盘中差异表达的长链非编码RNA（long non-coding RNA，LncRNA）及mRNA，并筛选有诊断标志物潜力的LncRNA。方法：从2018年9月~2020年12月于宁夏医科大学附属总医院脊柱骨科就诊的患者中选取STB患者（实验组）及脊柱退变患者（对照组）各28例。收集两组患者病变椎间盘并提取总RNA，两组各挑选3例合格标本通过Arraystar人类LncRNA V5.0版芯片进行检测并进行芯片数据处理计算各RNA的差异倍数（fold change，FC），其中FC≥2的LncRNA及mRNA为差异表达的RNA（P＜0.05）。将差异表达的LncRNA邻近的差异表达的mRNA视为cis-靶基因，将LncRNA可能结合的蛋白质视为trans-靶蛋白，并对mRNA、cis-靶基因、trans-靶蛋白进行基因功能注释分析。从差异表达的LncRNA中选取cis-靶基因与STB发病可能相关的LncRNA用剩余各25例标本进行RT-PCR验证，筛选有诊断标志物潜力的LncRNA并进行基因功能注释分析。结果：基于两组间芯片数据的差异共筛选出444个差异表达的LncRNA、186个差异表达的mRNA。基因功能注释分析提示差异表达的LncRNA的cis-靶基因主要参与对转录、翻译、剪接、免疫反应、自然杀伤细胞介导的细胞毒性等生物学过程的调控及信号通路的传递；trans-靶蛋白主要参与对RNA的剪接、加工、转运等基因表达过程的调控；差异表达的mRNA主要参与对转录、翻译、剪接、炎症反应、免疫反应等生物学过程的调控及信号通路的传递。RT-PCR验证发现LINC01128-201、Lnc-DNALI1-205在STB患者中存在明显下调表达，与芯片检测结果一致，且这两个LncRNA可能通过调控ISG15、SNIP1、HES4表达量在STB发病过程中发挥重要作用。结论：STB患者与脊柱退变患者椎间盘的LncRNA及mRNA存在较大的表达差异，其中下调表达的LINC01128-201、Lnc-DNALI1-205可能在STB发病过程中发挥重要作用，且有成为诊断标志物的潜力。

英文摘要:

  【Abstract】 Objectives: To detect the differentially expressed long non-coding RNA(LncRNA) and mRNA in intervertebral discs of patients with spinal tuberculosis(STB) by expression profile chip and filter LncRNA with the potential of becoming a diagnostic marker. Methods: 28 patients with STB(Test group) and 28 patients(Control group) with spinal degeneration were selected from the Department of Spinal Orthopaedics of General Hospital of Ningxia Medical University from September 2018 to December 2020. The diseased intervertebral discs of the two groups were collected and the total RNA was extracted. Three qualified samples from each group were detected by Arraystar Human LncRNA V5.0 chip and the fold change(FC) of each RNA was calculated by chip data processing. Among them, LncRNA and mRNA with FC≥2 were differentially expressed RNA(P<0.05). The differential mRNA adjacent to the differential LncRNA was regarded as the cis-target gene, and the protein that might be bound by LncRNA was regarded as the trans-target protein, and then the functions of mRNA, cis-target gene and trans-target protein were analyzed. The LncRNA whose cis-target gene may be related to the pathogenesis of STB was selected from the differential LncRNA and verified by RT-PCR in the remaining 25 samples to screen of LncRNA with the potential to become a diagnostic marker and then their functions were analyzed. Results: A total of 444 differential LncRNA and 186 differential mRNA were screened based on the difference of chip data between the two groups. The analysis of gene functional annotation showed that the cis-target genes of differential LncRNA were mainly involved in the regulation of biological processes such as transcription, translation, splicing, immune response, natural killer cell-mediated cytotoxicity and the transmission of signal pathways, while trans-target proteins were mainly involved in the regulation of gene expression processes such as RNA splicing, processing and transport. Differential mRNA was mainly involved in the regulation of transcription, translation, splicing, inflammation, immune response and other biological processes and the transmission of signal pathways. RT-PCR verification showed that there were significant down-regulated expressions of LINC01128-201 and Lnc-DNALI1-205 in STB patients, which were consistent with the results of microarray, and these two LncRNA may play an important role in the pathogenesis of STB by regulating the expression volume of ISG15, SNIP1 and HES4. Conclusions: There is a significant difference in the expression of LncRNA and mRNA between patients with STB and patients with spinal degeneration. The down-regulated expressions of LINC01128-201 and Lnc-DNALI1-205 may play an important role in the pathogenesis of STB and have the potential to become diagnostic markers.